Project summary Glial ensheathment of axons is a conserved feature of nervous systems that is essential for proper nervous system function. Impairment or loss of axonal wrapping underlies many debilitating conditions including multiple sclerosis, leukodystrophies, peripheral neuropathies, and CMT diseases. Despite many years of work our understanding of the molecular pathways that control glial development, glial-axon communication, and ensheathment of long axons, including myelination, is far from complete. Our understanding of non-myelinating forms of axon ensheathment is particularly sparse, despite the fact that the majority of peripheral axons (~70%) in humans are unmyelinated and encased by Remak Schwann cells. To address this gap in our understanding we propose to use the genetically tractable model Drosophila to characterize novel molecular mechanisms that promote glial ensheathment of axons and to study the functional roles of non-myelinating ensheathment in axon health and function in vivo. In Drosophila, specialized glia called wrapping glia (WG) ensheath peripheral axons in a manner closely resembling vertebrate Remak SCs. Recent studies (including our own preliminary data) have found that many genes that control the formation of vertebrate myelin also control axon ensheathment by WG in the fly, supporting strong molecular conservation between these forms of ensheathment. We have taken advantage of the fly to conduct a large-scale RNAi screen for novel regulators of ensheathment, and have identified a number of exciting new genes required for glial ensheathment of axons. One candidate to emerge from the screen, discoidin domain receptor (Ddr), encodes an evolutionarily conserved receptor tyrosine kinase activated by collagens. We show that loss of Ddr in WG results in profound defects in axonal ensheathment: although WG can grow longitudinally along the nerve they fail to insert processes between bundled axons to sort and ensheath them. Intriguingly, murine Ddr1 is highly expressed in oligodendrocytes and detailed expression profiling reveals that mDdr1 expression increases at the onset of wrapping during development and with the initiation of remyelination after injury, but functional roles for mDdr in ensheathment or myelination has not been investigated. Our preliminary work has also identified the Type XV/XVIII collagen homolog Multiplexin as required for axon ensheathment, possibly by acting as a ligand for Ddr. In Aim 1 we will characterize the role of Ddr in promoting axonal ensheathment, determine its autonomy of action, and perform a structure function analysis to define key aspects of Ddr signaling in vivo. In Aim 2 we will investigate the role of Mp in driving ensheathment and directly test our model that Mp acts in an autocrine fashion to activate the Ddr receptor on WG. Finally, in Aim 3 we will take advantage of the many genes identified in the screen that have mild to strong ensheathment defects to probe the function of non-myelinating ensheathment on neuronal health and physiology using behavioral assays and in vivo physiological studies. Our work will define the mechanistic basis of Ddr and Mp signaling during nerve assembly and glial ensheathment of axons, and help define the enigmatic but essential functions of non-myelinating forms of ensheathment in complex nervous systems.